EP1117428A2 - Compositions and methods for inhibiting angiogenesis - Google Patents

Abstract

The invention provides for methods of reducing or inhibiting angiogenesis, tumor growth and endothelial cell proliferation by the administration of compositions containing fragments, conformations, biological equivalent, or derivatives of antithrombin III. The invention also provides for pharmaceutical compositions comprising a fragment, conformation, biological equivalent, or derivative of antithrombin III and methods of identifying novel inhibitors of tumor growth, endothelial cell proliferation, and/or angiogenesis. The invention also relates to compositions and methods for altering angiogenesis in a mammal, as well as to methods of treatment for disorders associated with angiogenesis (e.g., cancer).

Description

COMPOSITIONS AND METHODS FOR INHIBITING ANGIOGENESIS

The invention was supported, in whole or in part, by grant CA45548 from the

National Institutes of Health. The Government has certain rights in the invention.

This application claims benefit of U.S. Provisional Applications Serial Nos.

60/103,526 filed October 8, 1998 and 60/116,131 filed January 15, 1999, the disclosures of

which are hereby incorporated by reference.

Blood vessels are constructed by two processes: vasculogenesis, whereby a primitive

vascular network is established during embryogenesis from multipotential mesenchymal

progenitors; and angiogenesis, in which preexisting vessels send out capillary sprouts to

produce new vessels. Endothelial cells are centrally involved in each process. They migrate,

proliferate and then assemble into tubes with tight cell-cell connections to contain the blood

(Hanahan, Science 277:48-50 (1997)). Angiogenesis occurs when enzymes, released by

endothelial cells, and leukocytes begin to erode the basement membrane, which surrounds the

endothelial cells, allowing the endothelial cells to protrude through the membrane. These

endothelial cells then begin to migrate in response to angiogenic stimuli, forming off-shoots

of the blood vessels, and continue to proliferate until the off-shoots merge with each other to

form the new vessels.

Normally angiogenesis occurs in humans and animals in a very limited set of

circumstances, such as embryonic development, wound healing, and formation of the corpus

luteum, endometrium and placenta. However, aberrant angiogenesis is associated with a

number of disorders, including, tumor metastasis. In fact, it is commonly believed that tumor

growth is dependent upon angiogenic processes. Thus, the ability to increase or decrease angiogenesis has significant implications for clinical situations, such as wound healing (e.g.,

graft survival) or cancer therapy, respectively.

Antithrombin or Antithrombin III (AT3) is a single chain glycoprotein involved in the

coagulation process. It is synthesized primarily in the liver with a signal peptide of 32 amino

acids necessary for its intracellular transport through the endoplasmic reticulum; the peptide

is then cleaved prior to secretion. Mourey et al, Biochimie 72:599-608 (1990).

AT3 is a member of the serpin family of proteins and functions as an inhibitor of

thrombin and other enzymes involved in the clotting cascade. As used herein, the active

native intact form of AT3 is designated the S (stressed) form (S-AT3). S-AT3 forms a tight

binding complex with thrombin (markedly enhanced by the presence of heparin) and other

enzymes (not all serpins have heparin affinity).

S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by a variety of

enzymes, including thrombin. Evans et al, Biochemistry 31:1262912642 (1992). For

example, it has been thought that thrombin binds to a reactive C-terminal loop of AT3 and

the resultant complex slowly dissociates releasing thrombin and cleaving off the C-terminal

loop of inactive AT3, resulting in R-AT3. R-AT3 is unable to bind thrombin and has a

conformation that is quite different from that of S-AT3. The role of R-AT3 had only been

known to facilitate hepatic clearance of the molecule.

Other forms of AT3, such as L-AT3, which is the group of forms of ATIII that

includes both the latent form and the locked form, are similar in conformation to R-AT3, and

are also known in the art. Carrell et al, Nature 353, 576-578 (1991); Wardell et al,

Biochemistry 36, 13133-13142 (1997). L-AT3, for example, can be produced by limited

denaturing and renaturing the AT3 protein under specific temperature conditions, e.g., with guanidium chloride.

Prior to the present invention, AT3 was not known to be associated with angiogenesis.

The present invention is, in one embodiment, drawn to a fragment, conformation,

derivative or biological equivalent of AT3 that inhibits endothelial cell proliferation,

angiogenesis and/or tumor growth in vivo.

In one embodiment, the invention relates to a method of inhibiting tumor growth by

delivering or administering a composition comprising a fragment, conformation, biological

equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation,

biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of

AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of

AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be

chosen from a synthesized fragment of AT3 that inhibits tumor growth, conformational

variations of other serpins that inhibit tumor growth, an aggregate form of AT3 that inhibits

tumor growth, or a fusion protein of AT3 that inhibits tumor growth. The composition may

further comprise a physiologically acceptable vehicle.

The invention further relates to a method of inhibiting endothelial cell proliferation

comprising delivering or administering a composition comprising a fragment, conformation,

biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment,

conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3,

the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the

R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3

may also be chosen from a synthesized fragment of AT3 that inhibits endothelial cell

proliferation, conformational variations of other serpins that inhibit endothelial cell proliferation, an aggregate form of AT3 that inhibits endothelial cell proliferation, or a fusion

protein of AT3 that inhibits endothelial cell proliferation. The composition may further

comprise a physiologically acceptable vehicle.

The invention also relates to a method of reducing or inhibiting angiogenesis

comprising delivering or administering a composition comprising a fragment, conformation,

biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment,

conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3,

the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the

R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3

may also be chosen from a synthesized fragment of AT3 that reduces angiogenesis,

conformational variations of other serpins that reduce angiogenesis, an aggregate form of

AT3 that reduces angiogenesis, or a fusion protein of AT3 that reduces angiogenesis. The

composition may further comprise a physiologically acceptable vehicle.

In another embodiment, the invention pertains to a method for identifying an inhibitor

of tumor growth or an agent that reduces tumor growth, comprising the steps of inoculating

an animal with an appropriate innoculum of tumor cells in each of two suitable inoculation

sites; identifying inhibition of growth of a tumor, known as the subordinate tumor, at one

inoculation site with concomitant growth of a tumor, known as the dominant tumor, at the

other inoculation site; isolating cells from the dominant tumor; and purifying a component

which inhibits endothelial cell proliferation and/or angiogenesis from the isolated cells. For

example, the component may be purified from conditioned media from the cells. In one

embodiment of the invention, the tumor cells are derived from tumors selected from the

group consisting of small cell lung cancers and hepatocellular carcinomas. In a particular embodiment, the inoculation sites are the flanks of the animal. In one embodiment the

inhibitor of tumor growth is an inhibitor of endothelial cell proliferation. In another

embodiment the inhibitor of tumor growth is an inhibitor of angiogenesis. In a further

embodiment of the invention, the method further comprises a step of selecting for an animal

in which inhibition of the growth of the subordinate tumor by the dominant tumor is

substantially complete.

The invention further relates to a method of inhibiting tumor growth comprising

delivering or administering an inhibitor of tumor growth identified by the methods described

herein to a mammal. In a preferred embodiment the inhibitor of tumor growth is a fragment,

conformation, biological equivalent, or derivative of AT3.

It is also within the practice of the invention to use a similar method to identify an

agent that reduces or an inhibitor of angiogenesis and/or endothelial cell proliferation. Such a

method would also comprise the steps of inoculating an animal with an appropriate inoculum

of tumor cells in each of two suitable inoculation sites; identifying inhibition of growth of a

tumor, known as the subordinate tumor, at one inoculation site with concomitant growth of a

tumor, known as the dominant tumor, at the other inoculation site; isolating cells from the

dominant tumor; and purifying a component which inhibits endothelial cell proliferation

and/or angiogenesis from the isolated cells. The component may be purified from

conditioned media from the cells and in a particular embodiment, the inoculation sites are the

flanks of the animal.

The invention further relates to a method of reducing or inhibiting angiogenesis

and/or endothelial cell proliferation comprising delivering or administering an inhibitor of

angiogenesis and/or endothelial cell proliferation identified by the methods described herein to a mammal. In a preferred embodiment the inhibitor of angiogenesis and/or endothelial cell

proliferation is a fragment, conformation, biological equivalent, or derivative of AT3.

The invention also relates to a method of treating a disorder mediated by angiogenesis

comprising delivering or administering a composition comprising a fragment, conformation,

biological equivalent, or derivative of AT3 in an amount effective to reduce angiogenesis to a

mammal. In a preferred embodiment the fragment, conformation, biological equivalent, or

derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that

include the active sites of the L form of AT3 and/or the R form of AT3. The fragment,

conformation, biological equivalent, or derivative of AT3 may also be chosen from a

synthesized fragment of AT3 that reduces angiogenesis, conformational variations of other

serpins that reduce angiogenesis, an aggregate form of AT3 that reduces angiogenesis, or a

fusion protein of AT3 that reduces angiogenesis. The composition may further comprise a

physiologically acceptable vehicle.

The invention also relates to a method of treating a disorder mediated by endothelial

cell proliferation comprising delivering or administering a composition comprising a

fragment, conformation, biological equivalent, or derivative of AT3 in an amount effective to

inhibit endothelial cell proliferation to a mammal. In a preferred embodiment the fragment,

conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3,

the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the

R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3

may also be chosen from a synthesized fragment of AT3 that inhibits endothelial cell

proliferation, conformational variations of other serpins that inhibit endothelial cell

proliferation, an aggregate form of AT3 that inhibits endothelial cell proliferation, or a fusion protein of AT3 that inhibits endothelial cell proliferation. The composition may further

comprise a physiologically acceptable vehicle.

The invention also relates to a method of enhancing angiogenesis comprising

delivering or administering a composition comprising an effective amount of an antagonist of

a fragment, conformation, biological equivalent, or derivative of AT3 wherein the fragment,

conformation, biological equivalent, or derivative of AT3 reduces angiogenesis to a mammal.

This method can be used, for example, in wound healing and assisted reproduction techniques

as well as in coronary artery surgery and the revascularization/collateralization of peripheral

vascular vessels. The composition may further comprise a physiologically acceptable

vehicle.

The invention also relates to a method of enhancing endothelial cell proliferation

comprising delivering or administering a composition comprising an effective amount of an

antagonist of a fragment, conformation, biological equivalent, or derivative of AT3 wherein

the fragment, conformation, biological equivalent, or derivative of AT3 inhibits endothelial

cell proliferation to a mammal. The composition may further comprise a physiologically

acceptable vehicle.

Another embodiment of the invention is a kit for detecting the presence of a fragment,

conformation, biological equivalent, or derivative of AT3. The kit may contain primary

reagents suitable for detecting the presence of the fragment, conformation, biological

equivalent, or derivative of AT3 and optional secondary agents suitable for detecting the

binding of the primary reagent to the fragment, conformation, biological equivalent, or

derivative of AT3. In a preferred embodiment, the fragment, conformation, biological

equivalent, or derivative of AT3 is the L form of bovine AT3, the R form of bovine AT3, the L form of human AT3, or the R form of human AT3.

The invention provides for direct administration of the fragment, conformation,

biological equivalent, or derivative of AT3, along with the use of the fragment, conformation,

biological equivalent, or derivative of AT3 with or without physiologically acceptable

vehicles, including but not limited to viral vectors including adenoviruses, lipids and any

other methods that have been employed in the art to effectuate delivery of biologically active

molecules.

The invention also provides for the production of a fragment, conformation,

biological equivalent, or derivative of AT3 in vivo by the delivery of an enzyme. It is also

within the practice of the invention to produce a fragment, conformation, biological

equivalent, or derivative of AT3 in vivo by the delivery of a composition that effectuates a

conformational change in a serpin.

The invention also relates to pharmaceutical compositions comprising a fragment,

conformation, biological equivalent, or derivative of AT3. The composition may be effective

for inhibiting tumor growth, angiogenesis, and/or endothelial cell proliferation. In one

embodiment, an anti-angiogenic pharmaceutical composition comprises a purified form of

AT3 that reduces angiogenesis. In a preferred embodiment the purified form of AT3 is the L

form or R form of AT3 or a fragment or sequence which includes the active site or region of

the L form or R form of AT3. The composition may further comprise a physiologically

acceptable vehicle. The fragment, conformation, biological equivalent, or derivative of AT3

may be an active ingredient in a pharmaceutical composition that includes carriers, fillers,

extenders, dispersants, creams, gels, solutions and other excipients that are common in the

pharmaceutical formulatory arts. The invention also provides for a method of delivering or administering a composition

comprising a fragment, conformation, biological equivalent, or derivative of AT3 by any

methods that have been employed in the art to effectuate delivery of biologically active

molecules, including but not limited to, administration of an aerosolized solution, intravenous

injection, orally, parenterally, topically, or transmucosally.

The invention also provides for a pharmaceutical composition that comprises

compositions to facilitate delivery of therapeutically effective amounts of the fragment,

conformation, biological equivalent, or derivative of AT3. The pharmaceutical compositions

of the invention may be formulated to contain one or more additional physiologically

acceptable substances that stabilize the compositions for storage and/or contribute to the

successful delivery of the fragment, conformation, biological equivalent, or derivative of

AT3.

Additional features and advantages of the invention will be set forth in the description

which follows, and, in part, will be apparent from the description, or may be learned by the

practice of the invention. The objectives and other advantages of the invention will be

realized and attained by the compounds and methods particularly pointed out in the written

description and claims hereof as well as the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la. shows three conformations of AT3. b. (SEQ ID NO: 1) shows the amino acid

sequence of human AT3 and the pancreatic elastase cleavage site of the c-terminal reactive

loop.

Figure 2 shows the sequence data and schematic of bovine AT3 and aaAT3. N-terminal

sequences were determined by automated Edman degradation on a PE/ABD Procise 494cLC

protein sequencer (Foster City, CA) with high sensitivity phenylthiohydantoin amino acid

detection by capillary HPLC. Sequence library searches and alignments were performed

against combined GenBank, Brookhaven Protein, SWISS-PROT, and PIR databases.

Figure 3 is a graph showing the effects of intact (S), cleaved (R) and (L) conformations of

bovine AT3 on capillary endothelial cell proliferation.

Figure 4 is a graph showing the effects of intact (S), cleaved (R) and (L) conformations of

human AT3 on capillary endothelial cell proliferation.

Figure 5 a. is a graph showing the results of treatment with human S-AT3, human R-AT3,

human L-AT3 and bovine R-AT3 in mice implanted with a human neuroblastoma. The mean

tumor volume and standard error for mice (n = 4/group) is shown. Tumor volume was

determined using the formula width² x length x 0.52. The experiment was terminated and

mice sacrificed and autopsied when control mice began to die or experience morbidity, b.

Photograph of representative mice from each group on the 12^th day of treatment. Arrows

indicate tumors that have partially regressed.

Figure 6 shows the treatment of human neuroblastoma with latent or locked human

antithrombin III. Mean tumor volume and standard error (n = 5/group).

Figure 7 depicts a model of concomitant resistance, a. By selective in vivo passage, a variant of NCI-H69; small cell lung cancer was developed in which a primary flank tumor

completely suppresses the growth of a second implant on the opposite flank (arrows), b. In

another variant of the small cell lung cancer line that does not produce aaAT3, no evidence of

concomitant resistance is observed.

Figure 8 Purification of aaAT3 from the conditioned media of NCI-H69i spheroids, a.

Purification scheme, b. SDS-PAGE (reduced) of intact AT3 (lane 1) and aaAT3 (lane 2)

purified from H69i conditioned media. The B-chain of the cleaved form is indicated with an

arrow.

DETAILED DESCRIPTION OF THE INVENTION

As previously discussed, aberrant angiogenesis is associated with a number of

disorders including tumor metastasis. Additionally, endothelial cells are associated with

angiogenesis. In the present invention, fragments, conformations, biological equivalents, or

derivatives of AT3 may exhibit anti-angiogenic and anti-tumor activity. For example, the R

and L forms of AT3 are endothelial cell specific. Thus, this invention provides for methods

of reducing or inhibiting angiogenesis, tumor growth and endothelial proliferations using

fragments, conformations, biological equivalents, or derivatives of AT3. The invention also

provides for methods of identifying inhibitors of tumor growth, endothelial cell proliferation,

and/or angiogenesis. The invention also provides for pharmaceutical compositions

comprising a fragment, conformation, biological equivalent, or derivative of AT3. The

elements of the invention will now be discussed.

AT3 can be derived from any organism which produces the protein in nature. In a

particular embodiment the organism is bovine or human. The amino acid sequence of bovine AT3 is available under GenBank Accession No. 1168462, and the amino acid sequence of

human AT3 is available under GenBank Accession No. 113936 (SEQ ID NO:l).

AT3 can be isolated from body fluids such as serum, ascites and urine. AT3 can also

be synthesized chemically or biologically, such as by cell culture or recombinant technology,

or produced transgenically. Similarly, the particular portions and conformations of AT3,

which are the subject of this invention, can be isolated from natural sources, produced

transgenically, or can be chemically or biologically synthesized, such as by guanidine

treatment or in vitro cleavage of AT3. Recombinant techniques known in the art include, but

are not limited to, gene amplification from DNA using polymerase chain reaction (PCR),

gene amplification from RNA using reverse transcriptase PCR and NASB A (nucleic acid

sequence based amplifications).

The particular portions and conformations of AT3 or its biological equivalents, which

are the subject of this invention, may also be produced by the use of an enzyme (e.g. elastase)

in vivo. For example, an enzyme may be used in vivo, with or without plasma or native AT3

to serve as an additional substrate, to produce a fragment, conformation, biological

equivalent, or derivative of AT3 that inhibits endothelial cell proliferation, angiogenesis

and/or tumor growth.

AT3 is a member of the serpin family of proteins and functions as an inhibitor of

thrombin and other enzymes involved in the clotting cascade. Serpins are a family of

proteins that function as serine protease inhibitors. As used herein, the active native intact

form of AT3 is designated the S (stressed) form (S-AT3). S-AT3 (Figure IA) is referred to

as the "stressed" form of AT3 due to the fact that it is a metastable conformation with the

reactive center loop (RCL) extended. This is the active form of AT3 in terms of inhibition of thrombin and other serine proteases.

S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by a variety of

enzymes, including thrombin. R-AT3 (Figure 1 A) is referred to as the "relaxed" form due to

the fact that the RCL has been cleaved by one of several proteases, including thrombin and

elastase. This results in the insertion of the N-terminal half of the loop as a sixth strand into

the A-beta sheet of AT3 to give a much more stable conformation than for the S-AT3 form.

This form is no longer active as an inhibitor of serine proteases. For example, thrombin binds

to a reactive C-terminal loop of AT3 and the resultant complex slowly dissociates releasing

thrombin and cleaving off the C-terminal loop of inactive AT3, resulting in R-AT3. In

particular, R-AT3 can be generated by enzyme cleavage of S-AT3 between Arg₃₉₃ and Ser₃₉₄

(human AT3 numbering). The amino acid sequence of human AT3 and the pancreatic

elastase cleavage site of the c-terminal reactive loop are shown in Figure IB. (For bovine R-

AT3, the cleavage site is between Ser₃₈₆ and Thre₃₈₇ as shown in Fig. 2.) The cleaved AT3

consists of disulfide-bonded A- and B-chains and is unable to bind thrombin. The cleavage

occurs spontaneously even at cold temperatures resulting in the R form of AT3. Enzymes

suitable for this cleavage include, but are not limited to, pancreatic elastase and thrombin. R-

AT3 is unable to inhibit thrombin and has a conformation that is quite different from that of

S-AT3. The role of R-AT3 had only been known to facilitate hepatic clearance of the

molecule.

L-AT3 (Figure IA) is a group of forms of AT3 that includes the both the latent form

and the locked form. These forms are structurally similar to the R-AT3 form in that all or

part of the N-terminal half of the RCL has been inserted as a sixth strand into the A-beta

sheet of AT3, resulting in a more stable conformation that is no longer active as a serine protease inhibitor. In the case of the "L- forms" however, there is no cleavage of the loop.

The latent conformation is a monomeric L-form, while the locked conformation also includes

dimers and oligomers, which are formed by insertion of the RCL from one AT3 molecule

into the A-beta sheet of another.

Surprisingly, it has been determined that certain conformations of AT3 reduce

angiogenesis, endothelial cell proliferation, and tumor growth. (As used herein, endothelial

cell proliferation also includes endothelial cell migration and tube formation.) For example,

R-AT3 has potent anti-angiogenic and anti-tumor activity which is not found in S-AT3.

Additionally, the stable locked and latent forms (L-AT3) of S-AT3, which are substantially

similar in conformation to R-AT3, also reduce or inhibit angiogenesis and tumor growth in

vivo and are endothelial cell specific in vitro. The invention relates to methods of inhibiting

endothelial cell proliferation, angiogenesis and/or tumor growth in a mammal comprising

delivering or administering to the mammal a composition comprising a fragment,

conformation, biological equivalent, or derivative of AT3, including but not limited to L-AT3

and R-AT3, and an optional physiologically acceptable vehicle. As described herein,

fragments conformations, derivatives and biological equivalents of AT3 include, but are not

limited to: other serpins and their conformational variations; fragments; conformations;

aggregate forms; and fusion proteins; which are active as inhibitors of angiogenesis,

endothelial cell proliferation and/or tumor growth. The invention also relates to a method of

treating a disorder mediated by angiogenesis or endothelial cell proliferation comprising

administering a composition comprising a fragment, conformation, derivative or biological

equivalent of AT3, including but not limited to L-AT3 and R-AT3, and an optional

physiologically acceptable vehicle to a mammal. The invention further relates to a method of treating cancer comprising administering a composition comprising an effective amount of a

fragment, conformation, derivative or biological equivalent of AT3, including but not limited

to L-AT3 and R-AT3, and an optional physiologically acceptable vehicle to a mammal.

The invention also relates to a method of enhancing angiogenesis or endothelial cell

proliferation comprising administering a composition comprising an effective amount of an

antagonist of AT3, e.g., an antagonist of S-AT3, an antagonist of R-AT3 or an antagonist of

L-AT3 to a mammal. For example, this method can be useful in the treatment of abnormal

ovulation, menstruation and placentation, and vasculogenesis, such as in tissue repair, wound

healing and tissue grafting.

The fragments, conformations, derivatives, and biological equivalents of AT3 having

anti-angiogenic properties, that inhibit the proliferation of endothelial cells, and/or have anti-

tumor activity are described herein. These AT3 fragments, conformations, derivatives and

biological equivalents are collectively termed herein "anti-angiogenic AT3 products," "anti-

proliferative AT3 products," aaAT, or aaAT3.

In addition to the sequences of AT3 described above, useful nucleic acid molecules

may comprise a nucleotide sequence which is greater than about 80 percent, preferably

greater than about 85 percent, more preferably greater than about 90 percent, and even more

preferably greater than about 95 percent, identical to the nucleotide sequences of S-AT3, R-

AT3 and L-AT3 deposited in GenBank. The substantially identical sequence should,

however, retain at least one of the activities of inhibition of endothelial cell proliferation,

inhibition of angiogenesis or inhibition of tumor growth (i.e., a biological equivalent).

To determine the percent identity of two nucleotide sequences, the sequences are

aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first nucleotide sequence). The nucleotides at corresponding nucleotide positions are then

compared. When a position in the first sequence is occupied by the same nucleotide as the

corresponding position in the second sequence, then the molecules are identical at that

position. The percent identity between the two sequences is a function of the number of

identical positions shared by the sequences (i.e., % identity = # of identical positions/total #

of positions x 100).

The determination of percent identity between two sequences can be accomplished

using a mathematical algorithm. A preferred, non-limiting example of a mathematical

algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al, Proc.

Mad. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the

NBLAST program which can be used to identify sequences having the desired identity to

nucleotide sequences of the invention. To obtain gapped alignments for comparison

purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res,

25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default

parameters of the respective programs (e.g., NBLAST) can be used. See

http://www.ncbl.nlm.nih.gov. In one embodiment, parameters for sequence comparison can

be set at W=12. Parameters can also be varied (e.g., W=5 or W=20). The value "W"

determines how many continuous nucleotides must be identical for the program to identify

two sequences as containing regions of identity.

As appropriate, nucleic acid molecules of the present invention can be RNA, for

example, mRNA, or DNA, such as cDNA and genomic DNA. DNA molecules can be

double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or

sense, strand or the non-coding, or antisense, strand. Preferably, the nucleic acid molecule comprises at least about 10 nucleotides, more preferably at least about 50 nucleotides, and

even more preferably at least about 200 nucleotides. The nucleic acid molecule can include

all or a portion of the coding sequence of a gene and can further comprise additional non-

coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory

sequences, for example). Additionally, the nucleic acid molecule can be fused to a marker

sequence, for example, a sequence which encodes a polypeptide to assist in isolation or

purification of the polypeptide. Such sequences include, but are not limited to, those which

encode a glutathione-S-transferase (GST) fusion protein and those which encode a

hernaglutin A (HA) polypeptide marker from influenza.

As used herein, an "isolated" gene or nucleic acid molecule is intended to mean a gene

or nucleic acid molecule which is not flanked by nucleic acid molecules which normally (in

nature) flank the gene or nucleic acid molecule (such as in genomic sequences) and/or has

been completely or partially purified from other transcribed sequences (as in a cDNA or RNA

library). For example, an isolated nucleic acid of the invention may be substantially isolated

with respect to the complex cellular milieu in which it naturally occurs. In some instances,

the isolated material will form part of a composition (for example, a crude extract containing

other substances), buffer system or, reagent mix. In other circumstance, the material may be

purified to essential homogeneity, for example as determined by PAGE or column

chromatography such as HPLC. Preferably, an isolated nucleic acid comprises at least about

50, 80 or 90 percent (on a molar basis) of all macromolecular species present. Thus, an

isolated gene or nucleic acid molecule can include a gene or nucleic acid molecule which is

synthesized chemically or by recombinant means. Recombinant DNA contained in a vector

is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as

partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA

transcripts of the DNA molecules of the present invention are also encompassed by "isolated"

nucleic acid molecules. Such isolated nucleic acid molecules are useful in the manufacture of

the encoded protein, as probes for isolating homologous sequences (e.g., from other

mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or

for detecting expression of the gene in tissue (e.g., human tissue) such as by Northern blot

analysis.

Thus, DNA molecules which comprise a sequence which is different from the

naturally-occurring nucleic acid molecule, but which, due to the degeneracy of the genetic

code, encode a substantially similar protein or polypeptide are useful in this invention. The

invention also encompasses variations of the nucleic acid molecules of the invention, such as

those encoding portions, analogues or derivatives of the encoded protein or polypeptide.

Such variations can be naturally-occurring, such as in the case of allelic variation, or non-

naturally-occurring, such as those induced by various mutagens and mutagenic processes.

Intended variations include, but are not limited to, addition, deletion and substitution of one

or more nucleotides which can result in conservative or non-conservative amino acid

changes, including additions and deletions. Preferably, the nucleotide variations are silent;

that is, they do not alter the characteristics or activity of the encoded protein or polypeptide

(i.e., a biological equivalent). As used herein, activities of the encoded protein or polypeptide

include, but are not limited to, inhibition of angiogenesis, inhibition of endothelial cell

proliferation and inhibition of tumor growth. The invention also encompasses sequences that

are not identical to AT3. The invention also pertains to nucleic acid molecules which hybridize under high

stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence

described herein. Hybridization probes are oligonucleotides which bind in a base-specific

manner to a complementary strand of nucleic acid. Such probes include polypeptide nucleic

acids, as described in Nielsen et al, Science 254, 14971500 (1991). Such nucleic acid

molecules can be detected and/or isolated by specific hybridization (e.g., under high

stringency conditions). "Stringency conditions" for hybridization is a term of art which refers

to the incubation and wash conditions, e.g., conditions of temperature and buffer

concentration, which permit hybridization of a particular nucleic acid to a second nucleic

acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the

first and second may share some degree of complementarity which is less than perfect (e.g.,

60%, 75%, 85%, 95%). For example, certain high stringency conditions can be used which

distinguish perfectly complementary nucleic acids from those of less complementarity.

"High stringency conditions", "moderate stringency conditions" and "low stringency

conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages

6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al, "Current Protocols

in Molecular Biology", John Wiley & Sons, (1998)) the teachings of which are hereby

incorporated by reference. Equivalent conditions can be determined by varying one or more

of the parameters given as an example, as known in the art, while maintaining a similar

degree of identity or similarity between the target nucleic acid molecule and the primer or

probe used. Hybridizable nucleic acid molecules are useful as probes and primers, e.g., for

diagnostic applications.

In addition to substantially full-length polypeptides encoded by nucleic acid molecules described herein, the present invention includes biologically active fragments of

the S-AT3, R-AT3 and L-AT3 biological equivalents, or analogs thereof, including organic

molecules which simulate the interactions of S-AT3, R-AT3 or L-AT3. Biologically active

fragments include any portion of the full-length polypeptide which confers a biological

function on the variant gene product, including ligand binding and antibody binding, and

particularly including inhibition of endothelial cell proliferation, angiogenesis or tumor

growth.

Also of use in the invention are fragments or portions of the isolated nucleic acid

molecules described above. The term "fragment" is intended to encompass a portion of a

nucleic acid molecule described herein which is from at least about 7 contiguous nucleotides

to at least about 25 contiguous nucleotides or longer in length. Such fragments are useful as

probes, e.g., for diagnostic methods, and also as primers. The nucleotide sequences may also

be an isolated portion of any of the nucleotide sequences of S-AT3, R-AT3 and L-AT3,

which portion is sufficient in length to distinctly characterize the sequence. Particularly

preferred primers and probes selectively hybridize to the nucleotide sequences of S-AT3, R-

AT3 and L-AT3 . For example, fragments which encode antigenic proteins or polypeptides

described herein are useful.

Also within the practice of the invention are anti-angiogenic AT3 products and anti-

proliferative AT3 products that are intended to encompass any fragments or conformations

(e.g., the L conformation) of AT3 which have anti-angiogenic and/or anti-pro liferative

activity, respectively. Anti-angiogenic activity and anti-proliferative activity can be assessed

according to methods described herein or according to other methods known in the art or may

be any fragments or biological equivalents that mimic the active site. The biological equivalents of AT3, may include, but are not limited to, fragments of

S-AT3, R-AT3, and L-AT3 that comprise the active site; synthetic compounds that mimic the

active site; conformational variations of other serpins; other conformations of AT3, aggregate

forms and fusion proteins that exhibit anti-angiogenic and anti-proliferative properties.

Conformational variations of other serpins that may be useful in the practice of the invention

include but are not limited to plasminogen activator inhibitor- 1 (PAI-1), ₂ antiplasmin, αl

proteinase inhibitor, heparin cofactor II, Cl inhibitor, αl antichymotrypsin, protease nexin 1,

and pigment epithelial derived factor.

This invention also pertains to an isolated protein or polypeptide encoded by the

nucleic acid molecules of the invention. The encoded proteins or polypeptides of the

invention can be partially or substantially purified (e.g., purified to homogeneity), and/or are

substantially free of other proteins. According to the invention, the amino acid sequence of

the polypeptide can be that of the naturally-occurring protein or can comprise alterations

therein. Such alterations include conservative or non-conservative amino acid substitutions,

additions and deletions of one or more amino acids; however, such alterations should

preserve at least one activity of the encoded protein or polypeptide, i.e., the altered or mutant

protein should be a biological equivalent of the naturally-occurring protein. The mutation(s)

should preferably preserve the endothelial cell proliferative inhibition, angiogenesis

inhibition or tumor growth inhibition activities of the native protein or polypeptide. The

presence or absence of biological activity or activities can be determined by various

functional assays as described herein. For example, glycosylation variants of AT3, along

with β-AT3 (Olson et al, Archives of Biochemistry and Biophysics 341(2): 212-221 (1997))

are within the scope of the biological equivalents of AT3. The β-AT3 form may also be useful as described herein.

Moreover, amino acids which are essential for the function of the encoded protein or

polypeptide can be identified by methods known in the art. Particularly useful methods

include identification of conserved amino acids in the family or subfamily, site-directed

mutagenesis and alanine-scanning mutagenesis (for example, Cunningham and Wells,

Science 244:1081-1085 (1989)), crystallization and nuclear magnetic resonance. The altered

polypeptides produced by these methods can be tested for particular biologic activities,

including immunogenicity and antigenicity.

Specifically, appropriate amino acid alterations can be made on the basis of several

criteria, including hydrophobicity, basic or acidic character, charge, polarity, size, the

presence or absence of a functional group (e.g., -SH or a glycosylation site), and aromatic

character. Assignment of various amino acids to similar groups based on the properties

above will be readily apparent to the skilled artisan; further appropriate amino acid changes

can also be found in Bowie et al, Science 247:1306-1310(1990).

For example, conservative amino acid replacements can be those that take place

within a family of amino acids that are related in their side chains. Genetically encoded

amino acids are generally divided into four families: (1) acidic=aspartate, glutamate; (2)

basic=lysine, arginine, histidine; (3) nonpolar — alanine, valine, leucine, isoleucine, proline,

phenylalanine, methionine, tryptophan; and (4) uncharged polar — glycine, asparagine,

glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are

sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect

that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a

glutamate, a threonine with a serine or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on activity or functionality.

The polypeptides of the present invention can be used to raise antibodies or to elicit

an immune response. The polypeptides can also be used as a reagent, e.g., a labeled reagent,

in assays to quantitatively determine levels of the protein or a molecule to which it binds

(e.g., a receptor or a ligand) in biological fluids. The polypeptides can also be used as

markers for tissues in which the corresponding protein is preferentially expressed, either

constitutively, during tissue differentiation, or in a diseased state. The polypeptides can be

used to isolate a corresponding binding partner, e.g., receptor or ligand, such as, for example,

in an interaction trap assay, and to screen for peptide or small molecule antagonists or

agonists.

The present invention also relates to antibodies which bind a polypeptide or protein of

the invention. For instance, polyclonal and monoclonal antibodies, including non-human and

human antibodies, humanized antibodies, chimeric antibodies and antigen-binding fragments

thereof (Current Protocols in Immunology, John Wiley & Sons, N.Y. (1994); EP Application

173,494 (Morrison); International Patent Application W086/01533 (Neuberger); and U.S.

Patent No. 5,225,539 (Winters)) which bind to the described S-AT3, R-AT3 or L-AT3

proteins or polypeptides are within the scope of the invention. A mammal, such as a mouse,

rat, hamster or rabbit, can be immunized with an immunogenic form of the protein (e.g., the

full length protein or a polypeptide comprising an antigenic fragment of the protein which is

capable of eliciting an antibody response). Techniques for conferring immunogenicity on a

protein or polypeptide include conjugation to carriers or other techniques well known in the

art. The protein or polypeptide can be administered in the presence of an adjuvant. The

progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen

to assess the levels of antibody.

As described herein, AT3 and/or a fragment, conformation, biological equivalent, or

derivative can be made or isolated by numerous methods known in the art, including, but not

limited to, purification, transgenic and recombinant methods.

The invention provides expression vectors containing a nucleic acid sequence

described herein, operably linked to at least one regulatory sequence. Many such vectors are

commercially available, and other suitable vectors can be readily prepared by the skilled

artisan. "Operably linked" or "operatively linked" is intended to mean that the nucleic acid

molecule is linked to a regulatory sequence in a manner which allows expression of the

nucleic acid sequence. Regulatory sequences are art recognized and are selected to produce

the encoded polypeptide or protein. Accordingly, the term "regulatory sequence" includes

promoters, enhancers, and other expression control elements which are described in Goeddel,

Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA

(1990). For example, the native regulatory sequences or regulatory sequences native to the

transformed host cell can be employed. It should be understood that the design of the

expression vector may depend on such factors as the choice of the host cell to be transformed

and/or the type of protein desired to be expressed. For instance, the polypeptides of the

present invention can be produced by ligating the cloned gene, or a portion thereof, into a

vector suitable for expression in either prokaryotic cells, eukaryotic cells or both (see, for

example, Broach, et al, Experimental Manipulation of Gene Expression, ed. M. Inouye

(Academic Press, 1983) p. 83; Molecular Cloning: A Laboratory Manual, 2nd Ed., ed.

Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17). Typically, expression constructs will contain one or more selectable markers, including, but

not limited to, the gene that encodes dihydrofolate reductase and the genes that confer

resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin and

streptomycin resistance.

Prokaryotic and eukaryotic host cells transfected by the described vectors are also

provided by this invention. For instance, cells which can be transfected with the vectors of

the present invention include, but are not limited to, bacterial cells such as E. coli (e.g., E

coli K12 strains), Streptomyces, Pseudomonas, Serratia marcescens and Salmonella

typhimurium, insect cells (baculovirus), including Drosophila, fungal cells, such as yeast

cells, plant cells and mammalian cells, such as thymocytes, Chinese hamster ovary cells

(CHO), and COS cells.

In one embodiment, at least one fragment, conformation, biological equivalent, or

derivative of AT3 that is useful in the practice of the invention is produced in vivo or ex vivo

via gene therapy. For example, gene therapy may be used to produce AT3 or a biological

equivalent. An enzyme that effectuates a conformational change in AT3 or a biological

equivalent to an anti-angiogenic product is then delivered to the AT3 or the biological

equivalent. Gene therapy may be used to produce an enzyme that effectuates a

conformational change in AT3 or a biological equivalent to an anti-angiogenic product, or

both an enzyme and AT3 or a biological equivalent. By using tissue specific expression an

anti-angiogenic product may be produced in vivo at a desired site.

Thus, a nucleic acid molecule described herein can be used to produce a recombinant

form of the protein via microbial or eukaryotic cellular processes. Ligating the polynucleic

acid molecule into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect, plant or mammalian) or

prokaryotic (bacterial cells), are standard procedures used in producing other well known

proteins. Similar procedures, or modifications thereof, can be employed to prepare

recombinant proteins according to the present invention by microbial means or tissue-culture

technology. Accordingly, the invention pertains to the production of encoded proteins or

polypeptides by recombinant technology.

The proteins and polypeptides of the present invention can be isolated or purified

(e.g., to homogeneity) from recombinant cell culture by a variety of processes. These

include, but are not limited to, anion or cation exchange chromatography, ethanol

precipitation, affinity chromatography and high performance liquid chromatography (HPLC).

The particular method used will depend upon the properties of the polypeptide and the

selection of the host cell; appropriate methods will be readily apparent to those skilled in the

art.

Following immunization, anti-peptide antisera can be obtained, and if desired,

polyclonal antibodies can be isolated from the serum. Monoclonal antibodies can also be

produced by standard techniques which are well known in the art (Kohler and Milstein,

Nature 256:495-497 (1975); Kozbar et al, Immunology Today 4:72 (1983); and Cole et al,

Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 7796 (1985)). The term

"antibody" as used herein is intended to include fragments thereof, such as Fab and F(ab)2.

Antibodies described herein can be used to inhibit the activity of the polypeptides and

proteins described herein, particularly in vitro and in cell extracts, using methods known in

the art. Additionally, such antibodies, in conjunction with a label, such as a radioactive label,

can be used to assay for the presence of the expressed protein in a cell from, e.g., a tissue sample, and can be used in an immunoabsorption process, such as an ELISA, to isolate the

protein or polypeptide. Tissue samples which can be assayed include human tissues, e.g.,

differentiated and non-differentiated cells, such as tumor cells. These antibodies are useful in

diagnostic assays, or as an active ingredient in a pharmaceutical composition. For example,

passive antibody therapy using antibodies which specifically bind S-AT3, R-AT3 or L-AT3

can be used to modulate (inhibit or enhance) endothelial cell proliferative- or angiogenic-

dependent processes such as reproduction, wound healing and tissue repair.

The present invention also encompasses the detection of conformations including but

not limited to S-AT3, R-AT3, and L-AT3, fragments, derivatives, conformational variations

of other seφins, and biological equivalents of AT3 in bodily fluids to determine the diagnosis

or prognosis of endothelial cell proliferation related or angiogenesis-related disorders. As

used herein, angiogenesis-related disorders include, but are not limited to, cancers, solid

tumors, blood born tumors such as leukemias, tumor metastasis, benign tumors such as

hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyogenic granulomas,

rheumatoid arthritis, psoriasis, ocular angiogenic diseases such as diabetic retinopathy,

retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular

glaucoma, retrolental fibroplasia and rubeosis, Osier- Webber Syndrome, myocardial

angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and

wound granulation. As used herein, endothelial cell proliferation-related disorders include,

but are not limited to, intestinal adhesions, atherosclerosis, scleroderma and hypertrophic

scars. Compounds described herein can also be used as birth control agents by preventing the

neovascularization required for embryo implantation.

The invention also relates to a kit for detecting the presence of fragments, conformations, derivatives, and biological equivalents of AT3, including S-AT3, R-AT3 or

L-AT3 in bodily fluids. Typically, the kit will comprise primary reagents (e.g., antibodies)

capable of detecting the presence of fragments, conformations, derivatives, and biological

equivalents in a sample. The kit may also comprise adjunct reagents suitable for detecting

binding of the primary reagent to the target.

The present invention also pertains to pharmaceutical compositions comprising

fragments, conformations, derivatives, and biological equivalents of AT3 including

polypeptides and other compounds described herein. For instance, a polypeptide or protein,

or prodrug thereof, of the present invention can be formulated with a physiologically

acceptable medium to prepare a pharmaceutical composition. In one embodiment, an anti-

angiogenic pharmaceutical composition comprises a purified form of AT3 that reduces

angiogenesis. In a preferred embodiment the purified form of AT3 is the L form or R form of

AT3 or a fragment or sequence which includes the active site or region of the L form or R

form of AT3. The particular physiological medium may include, but is not limited to, water,

buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and

dextrose solutions. The optimum concentration of the active ingredient(s) in the chosen

medium can be determined empirically, according to well known procedures, and will depend

on the ultimate pharmaceutical formulation desired. Formulation of an agent to be

administered will vary according to the route of administration selected (e.g., solution,

emulsion, capsule). An appropriate composition comprising the agent to be administered can

be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions,

suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or

suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed

oils, for instance. Intravenous vehicles can include various additives, preservatives, or fluid,

nutrient or electrolyte replenishers and the like (See, generally, Remington 's Pharmaceutical

Sciences, 17^th Edition, Mack Publishing Co., PA, 1985). For inhalation, the agent can be

solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer,

nebulizer or pressurized aerosol dispenser).

The pharmaceutical compositions of the present invention may also comprise a

composition that effectuates a conformational change in a serpin or produces a fragment,

conformation, derivative, and biological equivalent of AT3 in vivo, for example, by the

delivery of an enzyme.

Methods of introduction at the site of treatment include, but are not limited to,

intradermal, intramuscular, intra peritoneal, intravenous, rectal, vaginal, intra ocular, topical,

subcutaneous, oral and intra nasal. Other suitable methods of introduction can also include

gene therapy, rechargeable or biodegradable devices, viral vectors, naked DNA, lipids and

slow release polymeric devices. The pharmaceutical compositions of this invention can also

be administered as part of a combinatorial therapy with other agents. Nucleic acid sequences

of the invention can be used in gene therapy and introduced either in vivo or ex vivo into cells

for expression in a mammalian subject. Cells can also be cultured ex vivo in the presence of

proteins of the present invention in order to produce a desired effect on such cells. Treated

cells can then be introduced in vivo for therapeutic purposes.

The invention can be used to treat a variety of animals. Suitable animals as used

herein include mammals, including, but not limited to, primates (e.g., humans), dogs, cats,

cows, horses, pigs, sheep, goats and rodents (e.g., rats, mice and hamsters). Appropriate dosages (e.g., those containing an "effective amount") of a fragment, conformation, derivative

or biological equivalent of AT3 will depend upon the physical characteristics of the animal to

be treated and on the disorder and (progression thereof) to be treated. One of ordinary skill in

the art would readily be able to determine what would be an effective amount. The agent can

be administered alone or in combination with other agents or treatment regimes, including

chemotherapy and radiation. The agent can be administered in multiple or single

administrations provided sequentially or simultaneously.

It is also a subject of this invention to provide a new method for identifying an

inhibitor of tumor growth or an agent that reduces tumor growth. Prior methods typically

required that two separate tumor inoculations or implants be carried out, the second being

performed substantially later than the first. The reason for this approach was that it was

thought that if the second tumor implant occurred at the same time, or shortly after, the

primary tumor was implanted, the primary tumor would not be able to suppress the secondary

tumor, because by the time the primary tumor reached sufficient size to release the inhibitors

continuously into the circulation, angiogenesis in the secondary tumor would already be well

underway. Similarly, it was also believed that if two tumors were implanted simultaneously

(e.g., in opposite flanks), the inhibitors would have an equivalent inhibiting effect on each

other.

Work described herein has shown that tumor implants or inoculations can in fact be

carried out substantially simultaneously in an animal in an effort to identify inhibitors of

tumor growth. As used herein, "substantially simultaneously" means that the tumor implants

or inoculations occur at the same time or closely in sequence; there is no requirement that one

tumor be implanted or inoculated first and allowed to grow to a particular size. The method of this invention therefore provides a substantial reduction in the time necessary to carry out

the screening, as it is not necessary to wait for the first tumor to grow before implanting the

second.

According to the method of the invention, an animal (e.g., a mammal such as a

mouse, rat, guinea pig or primate) is inoculated with an appropriate inoculum of tumor cells

in each of two suitable inoculation sites substantially simultaneously. An appropriate

inoculum of tumor cells is an amount of tumor cells sufficient to cause formation of a tumor,

and is intended to include implantation of preformed tumors such as micrometastasis. The

tumor cells can be derived from any tumors; for example, the tumors can include, but are not

limited to, small cell lung cancers and hepatocellular carcinomas. Appropriate inoculation

sites include, but are not limited to, the subcutaneous space, the cornea, the lung, the breast,

the prostate, the testes and the brain. In a preferred embodiment, the inoculation sites are the

flanks of the animal.

The tumors are allowed to grow in the animal, and inhibition of growth of one tumor,

known as the subordinate tumor, with concomitant growth of the other tumor, known as the

dominant tumor, is identified. Tumor size can be measured using methods known in the art,

such as by measuring the diameter of the tumor using calipers. Using methods described

herein and known in the art, such as selective in vivo passaging, dominant tumors can be

selected which substantially completely inhibit the growth of the subordinate tumor. As used

herein, "substantially completely" is intended to include greater than about 80% inhibition of

growth of the subordinate tumor. In a preferred embodiment, the inhibition is greater than

about 90%, and in a particularly preferred embodiment, the inhibition is near 100%.

However, dominant tumors which inhibit the growth of the subordinate tumor to any degree are useful.

Once a suitable dominant tumor is identified, the component(s) which inhibits tumor

growth can be purified from the tumor. For example, the tumor can be grown in vitro and the

component can be purified from conditioned media from the tumor cells using methods

described herein or other methods known in the art. With respect to protein or polypeptide

identification, bands identified by gel analysis can be isolated and purified by HPLC, and the

resulting purified protein can be sequenced. Alternatively, the purified protein can be

enzymatically digested by methods known in the art to produce polypeptide fragments which

can be sequenced. The sequencing can be performed, for example, by the methods of Wilm

et al. Nature 379(6564):466-469 (1996). The protein may be isolated by conventional means

of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or

99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology

Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles

and Practice, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed), Guide to

Protein Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it

can be isolated from the supernatant in which the host cell is grown. If not secreted, the

protein can be isolated from a lysate of the host cells.

Potential inhibitors can be tested in endothelial cell proliferation assays and/or

angiogenesis assays (e.g., a CAM assay) to identify inhibitors of endothelial cell proliferation

and/or angiogenesis. For example, as described herein, bovine and human R-AT3 and L-AT3

were identified as inhibiting endothelial cell proliferation, angiogenesis and tumor growth.

Compounds identified by this method as inhibiting endothelial cell proliferation,

angiogenesis and/or tumor growth can be used in both in vitro and in vivo methods to inhibit endothelial cell proliferation, angiogenesis and/or tumor growth as described herein for AT3.

Furthermore, antagonists of identified compounds can be identified using art recognized

methods. For example, an antagonist to be tested can be combined with the compound in an

endothelial cell proliferation assay or angiogenesis assay, and the level of endothelial cell

proliferation or angiogenesis can be assessed relative to the results in the absence of the

putative antagonist. Antagonists can be nucleic acids, proteins or polypeptides, small

biologically active molecules, or large cellular structures and can be used to enhance

endothelial cell proliferation and/or angiogenesis, such as in wound healing. Use of

compounds identified by this method for inhibition or enhancement of endothelial cell

proliferation and/or angiogenesis and for inhibition of tumor growth, as well as novel

compounds identified by this method, are within the scope of the invention.

The present invention will now be illustrated by the following Examples, which are

not intended to be limiting in any way. The teachings of all references cited herein are

incoφorated herein by reference in their entirety.

EXAMPLES

Example 1: Purification of Bovine and Human Antithrombin III

Bovine calf serum was thawed and heat- inactivated (56C x 20 minutes) and then

stored at 4°C for 14-21 days to allow for degradation of AT3 to the R form. Serum was

diluted 3-fold with 10 mM Tris pH 7 and then applied to a CM Sepharose column (5 x 35

cm) coupled to a DEAE Sepharose column (5 x 35 cm) after equilibration with 10 mM Tris

pH 7. Both columns were washed extensively with 10 mM Tris pH 7 and then uncoupled.

The DEAE column was washed extensively with 50 mM NaCl in 10 m-M Tris and then

coupled to a heparin Sepharose column (2.5 x 3 5 cm) which was equilibrated with 0.2 M NaCl 10 mM Tris pH 7. Bound protein from the DEAE column was eluted directly onto the

heparin Sepharose column using 0.2 M NaCl 10 mM Tris pH 7 and the columns were

uncoupled. The heparin Sepharose column was washed extensively with 0.5 M NaCl and

then eluted with a continuous gradient of 0.6 - 2 M NaCl (550 ml total volume) followed by

an additional 250 ml of 2 M NaCl. Fractions were collected and an aliquot of each was tested

on capillary endothelial cells. Fractions that inhibited were pooled and concentrated using a

NanoSpin 30K centrifugal concentrator.

Human plasma was centrifuged (10,000 φm x 30 minutes), filtered (0.45 μm), and

diluted 3-fold with 10 mM Tris pH 7. The diluted plasma was then applied to a CM

Sepharose column (5 x 35 cm) coupled to a DEAE Sepharose column (5 x 35 cm) after

equilibration with 10 mM Tris pH 7. Both columns were washed extensively with 10 mM

Tris pH 7 and then uncoupled. The DEAE column was washed extensively with 50 mm

NaCl in 10 mM Tris and then coupled to a heparin Sepharose column (2.5 x 35 cm) which

were equilibrated with 0.2 M NaCl 10 mM Tris pH 7. Bound protein from the DEAE column

was eluted directly onto the heparin Sepharose column using 0.2 M NaCl 10 mM Tris pH 7

and the columns were uncoupled. The heparin Sepharose column was washed extensively

with PBS followed by 0.5 M NaCl in 10 mM Tris pH 7 and then eluted with a continuous

gradient of 0.6 - 2 M NaCl (550 ml total volume) followed by an additional 250 ml of 2 M

NaCl. Purified intact native AT3 was cleaved with porcine pancreatic elastase to produce the

R-conformation incubated at 4°C in 0.9 M guanidine and then dialyzed against PBS to

produce the L-conformation. Purity of the final samples were assessed by SDS-PAGE with

silver staining. Protein concentration was determined using a Biorad assay. Example 2: Production ofR and L forms of Antithrombin III

Intact bovine and human AT3 were obtained from Sigma or Calbiochem, respectively,

or from human plasma as described above. Human and bovine intact AT3 were incubated in

0.9 M guanidium chloride to produce the stable locked conformation (L form) of the

molecule as described previously by Carrell et al. After 12 hours of incubation, guanidium

chloride was removed by dialysis (30K membrane) in PBS and samples were concentrated.

Alternatively, AT3 was cleaved with pancreatic elastase as described. In order to obtain

complete cleavage, the method was modified and AT3 was incubated with elastase at a 1 :5

molar ratio for 12 hours at 37°C. The reaction was quenched by applying the mixture to a

heparin Sepharose column at 4°C. The cleaved AT3 was purified to homogeneity using

heparin Sepharose and then concentrated using a Nanospin 30K centrifugal concentrator.

Example 3: Inhibitory Activity on Endothelial Proliferation

To determine if the inhibitory activity on endothelial cell proliferation of AT3 was

specific, AT3 from bovine and human sources was screened on a panel of nonendothelial cell

lines in vitro, as specificity in vitro may predict for lack of toxicity in vivo. Of all the cell

types screened, only capillary endothelial cells were significantly inhibited even at log fold

higher doses. The same specificity was seen for the L- and R forms of human and bovine

AT3. The intact native (S-AT3) molecule had no significant effect on non-endothelial cells

and only marginally inhibited capillary endothelial cells at doses in excess of 10 ug/ml.

Purified intact bovine and human AT3 (S-AT3), R-AT3 and L-AT3 were tested on

capillary endothelial cells in a proliferation assay (Figures 3 and 4, respectively). The L-AT3

potently inhibited capillary endothelial cell proliferation in a dose dependent and reversible fashion with half maximal inhibition seen at approximately 50 ng/ml for both the bovine and

human protein (Figs. 3 and 4, respectively). The inhibition was comparable for that seen with

the cleaved form of AT3 from the H69 conditioned media or from a digestion of the intact

protein with pancreatic elastase. The intact native conformation of bovine and human AT3

had no effect on capillary endothelial cell proliferation at comparable doses (Figs. 3 and 4,

respectively) but did show marginal inhibition at doses in excess of 5 μg/ml. It was

determined that virtually all of the AT3 was in the cleaved (R) form that inhibited endothelial

cell proliferation in a dose dependent fashion (Figs. 3 and 4). The data demonstrates that the

conformational change that occurs after cleavage of the AT3 molecule confers antiangiogenic

activity. The term aaAT3 is used to describe the antiangiogenic form of AT3.

Example 4: Inhibition of in vivo Angiogenesis

In order to determine whether the AT3 fragment, R-conformation, or L-conformation

could inhibit in vivo angiogenesis, the chick chorioallantoic membrane (CAM) assay was

used. CAM Assay

Three-day-old, fertilized white Leghorn eggs (Spafas, Norwich, CT) were cracked,

and embryos with intact yolks were placed in 100 x 20 mm petri dishes. After three days of

incubation (37 °C and 3%CO₂), a methylcellulose disc containing AT3 was applied to the

CAM of individual embryos. The discs were made by desiccation of AT3 in 10 l of 0.45%

methylcellulose on teflon rods. After 48 hours of incubation, embryos and CAMs were

observed by means of a stereornicroscope. Embryos were observed daily until there was no

evidence of inhibitory zones.

Intact native AT3 had no effect on angiogenesis in the assay but did cause local bleeding at the injection site at higher doses. In contrast, both the R- and L forms of human

and bovine AT3 potently inhibited angiogenesis at doses of 20 μg per CAM without evidence

of bleeding. In all embryos tested with two separate batches of AT3, there was a potent and

sustained inhibition of angiogenesis. No hemorrhage was seen in any of the treated groups,

consistent with prior reports that demonstrate these conformations do not bind or inhibit

thrombin. There was no evidence of any toxic or inflammatory reaction from any of the

proteins tested in the assay at any dose.

Example 5: Inhibition of Tumor Growth

Treatment of Human Malignant Neuroblastoma

Male 6-8 week old C57BI6/J (Jackson Labs, Bar Harbor, ME) or SCID (MGH) mice

were used. Mice were acclimated, caged in groups of 4 or less, their backs shaved, and fed a

diet of animal chow and water ad libitum. Methoxyfurane by inhalation was used for

anesthesia and euthanasia.

Immunocompromised SCID mice were implanted with human neuroblastoma cells

and tumors were allowed to grow to 1% of body weight. This tumor line (SK-NAS) has a

consistent pattern of growth. Tumors were measured with a dial caliper, volumes determined

using the formula width 2 x length x 0.52, and the ratio of the treated-to-control volume was

determined for the last time point. When tumor volume reached 179-200 mm³, mice were

randomized into groups and treated with bovine cleaved AT3 (R) form or the cleaved (R),

locked (L) or native intact (S) conformations of human AT3 or vehicle control injected into

the subcutaneous flanks once daily at a site distant from the tumor at a dose of 25 mg/kg (500

μg per 20 gram mouse). The experiment was terminated and mice sacrificed and autopsied when control mice began to die or experience significant morbidity.

The intact native form of AT3 had no effect on tumor growth as compared to control

mice treated with vehicle alone (Fig. 5). In contrast, mice treated with the R- and L-

confoπnation of AT3 had a complete regression of the implanted neuroblastoma; tumors in

these mice persisted as small barely visible subcutaneous nodules (Fig. 5). No toxicity was

seen in any of the treated mice except for some local bleeding at the injection site of the mice

treated with the native intact AT3.

In a separate experiment, SCID mice implanted with human neuroblastoma were

treated with latent or locked human AT3 at a dose of 15 mg/kg/day (Fig. 6) and both potently

inhibited tumor growth. These data show that aaAT3 is a potent inhibitor of angiogenesis

and tumor growth.

Example 6: Method of Identifying Inhibitors of Angiogenesis, Endothelial Cell

Proliferation, and/ or Tumor Growth

In order for a carcinoma to expand beyond a microscopic prevascular state, it must

produce stimulators of angiogenesis in excess of inhibitors and must sustain this imbalance.

It has been demonstrated that the continued production of angiogenesis inhibitors provides a

mechanism for the inhibition of tumor growth by tumor mass. Using murine models of

concomitant resistance, angiostatin from a murine lung carcinoma and endostatin from a

murine hemangioendothelioma were identified. To determine if human tumors might also

produce inhibitors of angiogenesis, human small cell lung cancer was screened for the ability

of a primary tumor on the flank of an immunocompromised mouse to inhibit the growth of a

similar implant on the opposite flank. Small cell lung cancer was chosen because, clinically, metastatic small cell lung cancer often grows rapidly after definitive treatment of the primary

disease.

The phenomenon of the rapid growth of metastasis has been referred to as

concomitant immunity, which could be due to the production of an inhibitor of angiogenesis

by small cell lung cancer. Several human small cell lung cancer cell lines were screened for

the ability of a primary tumor on the flank of an immunocompromised mouse to inhibit the

growth of a similar tumor on the opposite flank. One of the tumor lines obtained, a variant of

NCI-H69 obtained from the ATCC, which was originally derived from a primary tumor,

inhibited a similar tumor by over 80%. By selective in vivo passage, two variants of the H69

line were developed (H69i and H69ni). A tumor model was developed using H69i in which

the inhibition of one tumor by another was virtually 100%) (line H69i) [Fig. 7A]. A second

variant was also developed in which one tumor did not inhibit the other to a significant

degree (H69ni) [Fig. 7B].

Tumor cell lines derived from the inhibitory and non-inhibitory variants of H69 were

established in vitro. To screen for evidence of the production of an angiogenesis inhibitor,

conditioned media was tested on bovine capillary endothelial cells in a 72 -hour proliferation

assay. When cells were nearly confluent, conditioned media was collected. Conditioned

media from the H69i cells potently and reversibly inhibited capillary endothelial cell

proliferation. Conditioned media from the H69ni cell line had no significant inhibitory effect

on the endothelial cells. The data demonstrates that the purified inhibitor of endothelial cell

proliferation generated by the H69i cells is at least in part responsible for the concomitant

resistance observed in the tumor model.

Collection of Conditioned Media and Cell Culture Human small cell lung carcinoma cell lines were obtained from the ATCC and

maintained in culture in DMEM supplemented with 10% heat-inactivated fetal calf serum and

1% glutamine-penicillin-streptomycin in a 37° C and 10% CO₂ incubator. Optimal

conditions were developed for conditioned media using the minimal volume of media

supplemented with the least amount of serum and the maximal contact time for cell viability.

To produce conditioned media, 80 milliliter of DMEM with 2.5% FCS and 1% GPS was

added to near confluent cells in 900 cm2 roller bottles. After 96 hours at 37 °C and 10% CO₂,

media was collected, centrifuged (10,000 φm for 20 minutes), filtered (0.45 μm), and stored

at 4°C. The cells were noted to grow as spheroids, which were loosely adherent to the

plastic. Media was collected every 96 hours until the spheroid density expanded beyond the

limits of the surface area of the roller bottle.

Purification of Inhibitory Activity from Conditioned Media

DEAE, CM, Iysine, and heparin Sepharose, Sephacryl S200 HR gel, and a

SynChropak RP-4 C4 reverse-phase column were all prepared according to the

manufacturers' recommendations. Pooled conditioned media (3 - 3.5 liters) was diluted three¬

fold with 10 mM Tris pH 7 and applied to a CM Sepharose column (5 x 35 cm) coupled to a

DEAE Sepharose column (5 x 35 cm) after equilibration with 10 mM Tris pH 7. Both

columns were washed extensively with 10 mM Tris pH 7 and then uncoupled. Each column

was eluted with a step gradient of NaCl in 10 mM Tris pH 7 with 50 mM, 0.2 M, 0.6 M, I M

and 2 M steps. The DEAE column was washed extensively with 50 mM NaCl in 10 mM Tris

to remove phenol red. Fractions with evidence of protein by A280 for each step were pooled

and an aliquot of each was applied to bovine capillary endothelial cells in a 72-hour

proliferation assay. The 0.2 M NaCl elution of the DEAE column was found to inhibit capillary endothelial cell proliferation and was diluted 2-fold with 10 mM Tris pH 7.

A heparin Sepharose column (2.5 x 35 cm) was equilibrated with 0.2 M NaCl 10 mM

Tris pH 7 and the inhibitory sample from the DEAE column was applied. The column was

washed with phosphate buffered saline. The column was then eluted with a continuous

gradient of 0.2-2 M NaCl (550 ml total volume) followed by an additional 250 ml of 2 M

NaCl. Fractions were collected and an aliquot of each was tested on capillary endothelial

cells. Inhibitory activity was found in fractions eluting at 1-1.2 M NaCl. Fractions that

inhibited were pooled and concentrated to 1.5 ml using a NanoSpin 30K centrifugal

concentrator.

The concentrated sample was applied to a Sephacryl S200 HR column (1.5 x 75 cm)

which was first equilibrated with PBS. The column was eluted with PBS and an aliquot of

each fraction collected was tested on capillary endothelial cells. Fractions with inhibitory

activity were pooled and concentrated to 1.5 ml using a NanoSpin centrifugal concentrator.

A SynChropak RP-4 (4.6 x 100 mm) high performance liquid chromatography

(HPLC) column was equilibrated with H₂0/0.1% trifluoroacetic acid (TFA) and HPLC-grade

reagents (Pierce, Rockford, IL) were used. The sample from gel filtration was filtered (0.22

μm) and then applied to the column and the column washed with the equilibration buffer.

The column was then eluted with a gradient of acetonitrile in 0.1 % TFA at 0.5 ml/min and I

ml fractions were collected. An aliquot of each fraction was evaporated by vacuum

centrifugation, resuspended in PBS, and applied to capillary endothelial cells. The inhibitory

activity was further purified to apparent homogeneity by subsequent cycles on the C4

column.

Fractions containing inhibitory activity evaluated by SDS-PAGE and the activity was associated with a band of apparent reduced molecular weight (Mr) of 55 kDa that

copurified with a 58-60 kDa band (Fig 8b). The inhibitory activity was associated with the

55 kDa band. The 55 kDa band was purified to homogeneity using a C4 reverse phase HPLC

column and eluted at 55% acetonitrile in 0.1 % trifluoroacetic acid (Fig. 8a).

The inhibitory fraction from the final HPLC run was analyzed by microsequence

analysis. Protein Microsequencing

The 55 kDa (reduced) inhibitor of capillary endothelial cell proliferation was purified

to homogeneity from batches of conditioned media. After the final HPLC, a sample

containing a single 55 kDa band after staining with silver was used for N-terminal sequence

analysis. The N-terminal sequence was determined by automated Edman degradation on a

PE/ABD Model 470A protein sequencer (Foster City, CA) operated with gas-phase delivery

of trifluoroacetic acid. Sequence library searches and alignments were performed against

combined GenBank, Brookhaven Protein, SWISS-PROT, and PIR databases. Searches were

performed at the National Center for Biotechnology Information through the use of the

BLAST network service.

Sequence analysis revealed identity to bovine AT3 (Fig. 2). Mass spectroscopy was

performed and revealed a molecular weight of 50 kd. These data identified the inhibitor of

angiogenesis as the cleaved form (R-conformation) of bovine AT3 (Fig. 2). SDS-PAGE

behavior is also data which tends to confirm the R confirmation. The cleavage site between

Ser₃₈₆ and Thre_3g7 for bovine AT3 has not previously been described and suggests that a novel

enzyme may be involved. Other enzymes that cleave AT3 include thrombin (Arg₃₉₄-Ser₃₉₅),

pancreatic elastase (Val_38g-Iso₃₈₉), human neutrophil elastase (Iso_39I-Ala₃₉₂), and a number of

others known in the art. Example 7: Purification of Bovine aaAT3 from BxPC3 Conditioned Media

BxPC3 conditioned media (5% FCS) was applied to heparin-Sepharose column,

previously equilibrated with 50 mM Tris-HCl, pH 7.4. The column was washed with 2-3

column volumes, and protein was eluted with incremental 0.5M NaCl steps (3 column

volumes). Fractions were assayed for the ability to inhibit the proliferation of endothelial

cells using well established assay techniques. The fraction eluting from heparin-Sepharose

between 1-1.5M NaCl contained a 58kDa protein that inhibited endothelial cell proliferation.

This fraction was concentrated by membrane filtration and applied to a Superdex200 gel

filtration column. Fractions were assayed for the ability to inhibit endothelial cell

proliferation, and a fraction containing a 58kDa protein was identified. Sequence analysis of

this single band determined it to be bovine antithrombin. Subsequent biochemcial analysis

indicated that this AT molecule, with anti-endothelial function, was in fact a "latent" form of

bovine AT produced specifically by the BxPC3 cells. This molecule inhibits the proliferation

and migration of endothelial cells in a dose dependent manner.

While this invention has been particularly shown and described with references to

preferred embodiments thereof, it will be understood by those skilled in the art that various

changes in form and details may be made therein without departing from the spirit and scope

of the invention as defined by the appended claims.

Claims

WE CLAIM:

1. A method of reducing angiogenesis in a mammal comprising delivering to said

mammal a composition comprising

at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III

wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III

reduces angiogenesis.

2. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

composition further comprises a physiologically acceptable vehicle.

3. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

chosen from

the L form of antithrombin III and the R form of antithrombin III.

4. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

chosen from

a synthesized fragment of antithrombin III that reduces angiogenesis,

a conformational variation of seφins that reduce angiogenesis,

an aggregate form of antithrombin III that reduces angiogenesis, and

a fusion protein of antithrombin III that reduces angiogenesis.

5. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

conformational variation of seφins is chosen from conformational variations of plasminogen

activator inhibitor- 1, α₂ antiplasmin, αl proteinase inhibitor, heparin cofactor II, Cl inhibitor, αl antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.

6. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

produced transgenically, recombinantly, or purified from mammal antithrombin III.

7. A method of reducing angiogenesis in a mammal according to Claim 1 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

produced in vivo by the delivery of an enzyme.

8. A method of reducing endothelial cell proliferation in a mammal comprising

delivering to said mammal a composition comprising

at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III

wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III

reduces endothelial cell proliferation.

9. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said composition further comprises a physiologically acceptable vehicle.

10. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is chosen from

the L form of antithrombin III and the R form of antithrombin III.

11. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is chosen from

a synthesized fragment of antithrombin III that reduces endothelial cell proliferation, a conformational variation of seφins that reduce endothelial cell proliferation,

an aggregate form of antithrombin III that reduces endothelial cell proliferation, and

a fusion protein of antithrombin III that reduces endothelial cell proliferation.

12. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said conformational variation of seφins is chosen from conformational variations of

plasminogen activator inhibitor- 1, α₂ antiplasmin, αl proteinase inhibitor, heparin cofactor II,

Cl inhibitor, αl antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.

13. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is produced transgenically, recombinantly, or purified from mammal

antithrombin III.

14. A method of reducing endothelial cell proliferation in a mammal according to Claim 8

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is produced in vivo by the delivery an enzyme.

15. A method of reducing tumor growth in a mammal comprising delivering to said

mammal a composition comprising

at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III

wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III

reduces tumor growth.

16. A method of reducing tumor growth in a mammal according to Claim 15 wherein said

composition further comprises a physiologically acceptable vehicle.

17. A method of reducing tumor growth in a mammal according to Claim 15 wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

chosen from

the L form of antithrombin III and the R form of antithrombin III.

18. A method of reducing tumor growth in a mammal according to Claim 15 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

chosen from

a synthesized fragment of antithrombin III that reduces endothelial cell proliferation,

a conformational variation of seφins that reduce endothelial cell proliferation,

an aggregate form of antithrombin III that reduces endothelial cell proliferation, and

a fusion protein of antithrombin III that reduces endothelial cell proliferation.

19. A method of reducing tumor growth in a mammal according to Claim 15 wherein said

conformational variation of seφins is chosen from conformational variations of plasminogen

activator inhibitor- 1, α₂ antiplasmin, αl proteinase inhibitor, heparin cofactor II, Cl inhibitor,

αl antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.

20. A method of reducing tumor growth in a mammal according to Claim 15 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III

is produced transgenically, recombinantly, or purified from mammal antithrombin III.

21. A method of reducing tumor growth in a mammal according to Claim 15 wherein said

at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is

produced in vivo by the delivery of an enzyme.

22. A method for identifying an agent that reduces tumor growth or angiogenesis

comprising the steps of

a) inoculating a mammal with an inoculum of tumor cells in each of two inoculation sites substantially simultaneously;

b) identifying reduction of growth of a tumor at one inoculation site, wherein

said tumor at the one inoculation site is a subordinate tumor,

with concomitant growth of a tumor at the other inoculation site, wherein said

tumor at the other inoculation site is a dominant tumor;

c) isolating cells from said dominant tumor; and

d) purifying a component which reduces endothelial cell proliferation,

angiogenesis

or both endothelial cell proliferation and angiogenesis from the isolated

dominant tumor cells.

23. A method for identifying an agent that reduces tumor growth or angiogenesis

according to Claim 22 wherein the tumor cells are derived from tumors selected from small

cell lung cancers and hepatocellular carcinomas.

24. A method for identifying an agent that reduces tumor growth or angiogenesis

according to Claim 22 wherein the inoculation sites are the flanks of the mammal.

25. A method for identifying an agent that reduces tumor growth or angiogenesis

according to Claim 22 wherein the component of step (d) is purified from conditioned media

from the cells of step (c).

26. A method for identifying an agent that reduces tumor growth or angiogenesis

according to Claim 22 wherein the agent that reduces tumor growth reduces endothelial cell

proliferation.

27. A method for identifying an agent that reduces tumor growth or angiogenesis

according to Claim 22 wherein the agent that reduces tumor growth reduces angiogenesis.

28. A method of reducing tumor growth comprising delivering to a mammal the agent

that reduces tumor growth or angiogenesis identified by the method according to Claim 22.

29. A method of treating a disorder mediated by endothelial cell proliferation comprising

delivering to a mammal a composition comprising at least one fragment, conformation,

biological equivalent, or derivative of antithrombin III wherein said fragment, conformation,

biological equivalent, or derivative of antithrombin III is delivered in an amount effective to

reduce endothelial cell proliferation.

30. A method of treating a disorder mediated by endothelial cell proliferation according

to Claim 29 wherein said at least one fragment, conformation, biological equivalent, or

derivative of antithrombin III is chosen from

the L form of antithrombin III and the R form of antithrombin III.

31. A method of treating a disorder mediated by endothelial cell proliferation according

to Claim 29 wherein said at least one fragment, conformation, biological equivalent, or

derivative of antithrombin III is chosen from

a synthesized fragment of antithrombin III that reduces endothelial cell proliferation,

a conformational variations of seφins that reduce endothelial cell proliferation,

an aggregate form of antithrombin III that reduces endothelial cell proliferation, and

a fusion protein of antithrombin III that reduces endothelial cell proliferation.

32. A method of treating a disorder mediated by endothelial cell proliferation according

to Claim 29 wherein said conformational variation of seφins is chosen from conformational

variations of plasminogen activator inhibitor- 1, α₂ antiplasmin, l proteinase inhibitor,

heparin cofactor II, Cl inhibitor, αl antichymotrypsin, protease nexin 1 or pigment epithelial

derived factor.

33. A method of treating a disorder mediated by endothelial cell proliferation according

to Claim 29 wherein said at least one fragment, conformation, biological equivalent, or

derivative of antithrombin III is produced transgenically, recombinantly, or purified from

mammal antithrombin III.

34. A method of treating a disorder mediated by angiogenesis comprising delivering to a

mammal a composition comprising at least one fragment, conformation, biological

equivalent, or derivative of antithrombin III wherein said fragment, conformation, biological

equivalent, or derivative of antithrombin III is delivered in an amount effective to reduce

angiogenesis.

35. A method of treating a disorder mediated by angiogenesis according to Claim 34

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is chosen from

the L form of antithrombin III and the R form of antithrombin III.

36. A method of treating a disorder mediated by angiogenesis according to Claim 34

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is chosen from

a synthesized fragment of antithrombin III that reduces angiogenesis,

a conformational variation of seφins that reduce angiogenesis,

an aggregate form of antithrombin III that reduces angiogenesis, and

a fusion protein of antithrombin III that reduces angiogenesis.

37. A method of treating a disorder mediated by angiogenesis according to Claim 34

wherein said conformational variation of seφins is chosen from conformational variations of

plasminogen activator inhibitor- 1, α₂ antiplasmin, αl proteinase inhibitor, heparin cofactor II, Cl inhibitor, αl antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.

38. A method of treating a disorder mediated by angiogenesis according to Claim 35

wherein said at least one fragment, conformation, biological equivalent, or derivative of

antithrombin III is produced transgenically, recombinantly, or purified from mammal

antithrombin III.

39. A method of enhancing angiogenesis or endothelial cell proliferation comprising

delivering a composition comprising at least one antagonist of a fragment, conformation,

biological equivalent, or derivative of antithrombin III to a mammal wherein said fragment,

conformation, biological equivalent, or derivative of antithrombin III reduces angiogenesis.

40. A anti-angiogenic pharmaceutical composition comprising

a purified form of antithrombin III that reduces angiogenesis chosen from

the (R) form of antithrombin III or the (L) form of antithrombin III, and

a physiologically acceptable carrier.